The ability of an enzyme to discriminate among many potential substrates is an important factor in maintaining the fidelity of most biological functions. While substrate selection can be regulated on many levels in a biological context, such as spatial and temporal localization of enzyme and substrate, concentrations of enzyme and substrate, and requirement of cofactors, the substrate specificity at the enzyme active site is the overriding principle that determines the turnover of a substrate. Characterization of the substrate specificity of an enzyme clearly provides invaluable information for the dissection of complex biological pathways. Definition of substrate specificity also provides the basis for the design of selective substrates and inhibitors to study enzyme activity.
Of the genomes that have been completely sequenced, 2% of the gene products encode proteases (Barrett, A. J., et al., (1998) Handbook of Proteolytic Enzymes (Academic Press, London)). This family of enzymes is crucial to every aspect of life and death of an organism. With the identification of new proteases, there is a need for the development of rapid and general methods to determine protease substrate specificity. While several biological methods, such as peptides displayed on filamentous phage (Matthews, D. J., et al. (1993) Science 260:1113-7; Ding, L., et al., (1995) Proceedings of the National Academy of Sciences of the United States of America 92:7627-31), and chemical methods, such as support-bound combinatorial libraries (Lam, K. S., et al., (1998) Methods in Molecular Biology, 87:1-6), have been developed to identify proteolytic substrate specificity, few offer the ability to rapidly and continuously monitor proteolytic activity against complex mixtures of substrates in solution.
The use of 7-amino-4-methyl coumarin (AMC) fluorogenic peptide substrates is a well-established method for the determination of protease specificity (Zimmerman, M., et al., (1977) Analytical Biochemistry 78:47-51). Specific cleavage of the anilide bond liberates the fluorogenic AMC leaving group allowing for the simple determination of cleavage rates for individual substrates. More recently, arrays (Lee, D., et al., (1999) Bioorganic and Medicinal Chemistry Letters 9:1667-72) and positional-scanning libraries (Rano, T. A., et aL, (1997) Chemistry and Biology 4:149-55) of AMC peptide substrate libraries have been employed to rapidly profile the N-terminal specificity of proteases by sampling a wide range of substrates in a single experiment. Each of these published efforts was designed for profiling caspases, cysteine proteases that require an Asp residue at the P1-position for substrate turnover. This requirement allows for the convenient attachment of the P1-Asp to the solid-support through the carboxylic acid side-chain. Since most proteases do not require P1-Asp/Glu for activity, libraries generated by these methods have limited applicability. Naturally, fluorogenic substrates that contain P1-amino acids that do not possess adequate side-chain functionality for attachment to a solid support in a straightforward manner (Gly, Leu, Val, Ile, Ala, Pro, Phe) will not be amenable to similar synthetic strategies.
Recently Fmoc-based synthesis methods to displace support-bound peptides with nucleophiles in a final cleavage step to produce C-terminal modified peptides have been developed (Backes et al., (1999) Journal of Organic Chemistry 64:2322-2330). The preparation of fluorogenic peptide substrates with any residue at the P1-position is possible by the preparation of AMC-amino acid derivatives, which are then used as nucleophiles to produce the AMC-peptide substrates (Backes et al. (2000) Nature Biotechnology 18(2): 187-193).
Support bound fluorogenic materials are also known in the art. For example, Adamczyk et al., Bioorg. Med. Chem. Lett., 9:217-220 (1999), have disclosed resin-supported fluorophores prepared from a new N-hydroxysuccinimidyl resin. The resin-bound active esters were used to prepare conjugates with haptens, such as estriol, thyroxine, phenytoin, etc. As the fluorophore is transferred from the resin to the free hapten, the resin-bound fluorophores of Adamczyk et al. do not constitute an appropriate starting point for the solid-phase synthesis of a peptide, nor is the use of the resin-bound fluorophore for derivatization of pre-formed peptides disclosed.
While the art provides a selection of methods that are useful for labeling materials with fluorophores, a method for the solid-phase synthesis of fluorogenic peptides, which begins with a resin-bound fluorophore, and materials that allow the method to be practiced, would represent a significant advance in the art. Such a method has great utility and provides a general strategy for the preparation of fluorogenic peptide substrate libraries. An innovative method would meet the following objectives: (1) the solid-phase synthesis method should enable direct incorporation of at least all 20 proteinogenic amino acids at every position, including the P1-position; (2) the method should be compatible with art-recognized solid-phase peptide synthesis protocols and instrumentation; and (3) the method should be flexible enough to enable the rapid synthesis of any single substrate, substrate array, and positional scanning library. Quite surprisingly, the present invention provides such a method.
The present invention provides, for the first time, a highly efficient method for the preparation of fluorogenic compound libraries, particularly peptide substrate libraries based upon a new bifunctional fluorogenic-leaving group. The leaving group of the invention is exemplified by 7-amino-4-carbamoylmethyl-coumarin (ACC). In an illustrative embodiment, using Fmoc-synthesis protocols, all 20 proteinogenic amino acids can be directly coupled to the support bound ACC-leaving group to provide general sets of substrates for analyzing protease substrate specificity. The versatility of the solid-phase synthesis strategy allows for substrate-arrays (Lee, D., et al., (1999) Bioorganic and Medicinal Chemistry Letters 9:1667-72) and positional scanning libraries (Rano, T. A., et aL, (1997) Chemistry and Biology 4:149-55) of any configuration to be rapidly prepared. The substrate specificity of numerous representative serine and cysteine proteases were profiled to show the utility and generality of libraries generated by the ACC method.
Thus, in a first aspect, the present invention provides a material having the structure: 
wherein: R1, R2, R3, R4, R5 and R6 are members independently selected from the group consisting of H, halogen, xe2x80x94NO2, xe2x80x94CN, xe2x80x94C(O)mR7, xe2x80x94C(O)NR8R9, xe2x80x94S(O)tR10, xe2x80x94SO2NR11R12, xe2x80x94OR13, substituted or unsubstituted alkyl, xe2x80x94R14xe2x80x94SS, and xe2x80x94NHR15 with the proviso that at least one of R1, R2, R3, R4, R5 and R6 is xe2x80x94R14xe2x80x94SS and at least one of R1, R2, R3, R4, R5 and R6 is xe2x80x94NHR15. R7, R8, R9, R10, R11, R12 and R13 are members independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl. R14 is a linking group adjoining the fluorogenic moiety and the solid support. R15 is a member selected from the group consisting of amine protecting groups, xe2x80x94C(O)-AA and xe2x80x94C(O)xe2x80x94P. P is a peptide sequence. AA is an amino acid residue. The subscript m is a member selected from the group consisting of the integers 1 and 2. The subscript t is a member selected from the group consisting of the integers from 0 to 2; and SS is a solid support.
In a second aspect, the present invention provides a fluorogenic peptide comprising a fluorogenic moiety covalently bound to a peptide sequence. The peptide includes the structure:
Rxe2x80x94Pxe2x80x83xe2x80x83(VII) 
wherein, P is a peptide sequence having a structure that is substantially identical to that set forth in Formula II. R is a fluorogenic moiety having a structure substantially similar to the fluorogenic moiety of Formula I. The fluorogenic group substituents, R1, R2, R3, R4, R5 and R6, are members independently selected from the group consisting of H, halogen, xe2x80x94NO2, xe2x80x94CN, xe2x80x94C(O)mR7, xe2x80x94C(O)NR8R9, xe2x80x94S(O)tR10, xe2x80x94SO2NR11R12, xe2x80x94OR13, substituted or unsubstituted alkyl, xe2x80x94NHC(O)xe2x80x94P, and xe2x80x94R20xe2x80x94Y. At least one of R1, R2, R3, R4, R5 and R6 is xe2x80x94R20xe2x80x94Y and at least one of R1, R2, R3, R4, R5 and R6 is xe2x80x94NHC(O)xe2x80x94P. R7, R8, R9, R10, R11, R12 and R13 are members independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl. R20 is either present or absent, and when present, is a member selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; when R20 is absent, Y is attached directly to the fluorogenic moiety. Y is an organic functional group or methyl, and is preferably a member selected from the group consisting of xe2x80x94COOR17R21, CONR17R21, xe2x80x94C(O)R17, xe2x80x94OR17, xe2x80x94SR17, xe2x80x94NR17R21, xe2x80x94C(O)NR17R21, and xe2x80x94C(O)SR17. R17 and R21 are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl. The subscript m is a member selected from the group consisting of the integers 1 and 2; and t is a member selected from the group consisting of the integers from 0 to 2.
In a further aspect, the present invention provides a library of fluorogenic peptides having a structure according to Formula VII. The library includes at least a first peptide having a first peptide sequence covalently attached to a first fluorogenic moiety and a second peptide having a second peptide sequence covalently attached to a second fluorogenic moiety. For each of each of the peptides of the library, P is independently selected from peptide sequences, preferably having the structure:
xe2x80x94C(O)-AA1-AA2-(AAi)J-2)xe2x80x83xe2x80x83(II). 
Each of AA1 through AAi is an amino acid residue which is a member independently selected from the group consisting of natural amino acid residues, unnatural amino acid residues and modified amino acid residues. Each J is independently selected and denotes the number of amino acid residues forming the first peptide sequence and the second peptide sequence and is a member selected from the group consisting of the numbers from 1 to 10. J can have the same value for each of the peptide sequences in a particular library, or it can have a different value for two or more of the peptides of the library. Each i is independently selected and denotes the position of the amino acid residue relative to AA1 and when J is greater than 2, i is a member selected from the group consisting of the numbers from 3 to 10.
For each of the peptides of the library, R is independently selected from fluorogenic moieties having a structure according to Formula I. Thus, the fluorogenic group(s) can be the same for each of the peptides of a particular library or the structure of R can vary in a selected manner for two or more peptides of the library.
For each of the library peptides having a structure according to Formula I, the substituents of the fluorogenic group, R1, R2, R3, R4, R5, and R6 are independently selected from the group consisting of H, halogen, xe2x80x94NO2, xe2x80x94CN, xe2x80x94C(O)mR7, xe2x80x94C(O)NR8R9, xe2x80x94S(O)tR10, xe2x80x94SO2NR11R12, xe2x80x94OR13, substituted or unsubstituted alkyl, xe2x80x94NHxe2x80x94C(O)-P, R20xe2x80x94Y, and xe2x80x94R4xe2x80x94SS. For each library peptide, at least one of R1, R2, R3, R4, R5, and R6 is a member independently selected from xe2x80x94R14xe2x80x94SS and xe2x80x94R14xe2x80x94Y and at least one of R1, R2, R3, R4, R5, and R6 is xe2x80x94NHxe2x80x94C(O)xe2x80x94P. R7, R8, R9, R10, R11, R12 and R13 for each library peptide are members independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl. R14 is a linking group adjoining the fluorogenic moiety and the solid support. R20 is either present or absent, and when present, is a member selected from the group consisting of substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl; when R20 is absent, Y is attached directly to the fluorogenic moiety. The subscript m is a member selected from the group consisting of the integers from 1 to 2. The subscript t is a member selected from the group consisting of the integers from 0 to 2. Y is an organic functional group or methyl, and is preferably a member selected from the group consisting of xe2x80x94COOR17, CONR17R21, xe2x80x94C(O)R17, xe2x80x94OR17, xe2x80x94SR17, NR17R21, xe2x80x94C(O)NR17R21, and xe2x80x94C(O)SR17. For each library peptide, R17 and R21 are members independently selected from the group consisting of H and substituted or unsubstituted alkyl. SS is a solid support.
Other objects and advantages of the present invention will be apparent from the Detailed Description, which follows.